The overall goals of this research are first, determination of the drug binding motifs in the regulatory domains of the proteins involved in resistance and second, elucidation of the molecular mechanisms of an ion pump whose genes are natural components of arsenical resistances. The clinically isolated resistance plasmid R773 carries the arsenical resistance (ars) operon that encodes an ATP-coupled extrusion pump for arsenite (AsO2-) and antimonite (SbO2-). The operon has five genes. The arsR and arsD genes encode repressors. The arsA gene product is an ATPase that serves as the catalytic subunit of the pump, and the arsB gene product is the membrane subunit of the pump. ArsC is an arsenate reductase that converts As(V) to As(III), the substrate of the pump, thus expanding the range of resistance. Goals of the projects include determination of As(III) binding sites by MALDI-MS and peptide mapping of the As(III)-protein complexes. ArsR binds As(III) tightly in a three-coordinate structure with three cysteine thiolates. ArsR is a dimer that could potentially bind one or two As(III). The stoichiometry of binding will be determined by mass spectroscopy. ArsD contains three vicinal cysteine pairs. Each of these is a potential As(III) binding site. The stoichiometry of As(III) binding to ArsD also can be established by mass spectroscopy. Mutants lacking each cysteine will be analyzed.